Intracellular estradiol binding protein

ABSTRACT

Described herein is a novel intracellular estradiol binding protein (“IEBP”), as well as a polynucleotide encoding this protein and various cells and cell lines producing and/or overexpressing it. IEBP is believed to play a role in the modulation of estrogen signaling and in the physiological resistance to the same. Abnormally elevated or decreased levels of IEBP may thus be a component of the etiology of diseases generally correlated with estrogen signaling, such as, by way of example, breast cancer and osteoporosis. Various embodiments of the present invention are believed to provide important tools for developing treatments for these conditions, such as, for example, by providing means for screening therapeutic compounds and identifying a genetic target for therapy.

This application claims the benefit of priority under 35 U.S.C. § 119 ofprovisional application Ser. No. 60/468,717, filed May 7, 2003, thecontents of which are hereby incorporated by reference.

GOVERNMENT RIGHTS

The invention described herein arose in the course of or under Grant No.1 RO1 DK55843-01A1 between the National Institutes of Health and theDivision of Endocrinology and Metabolism at Cedars-Sinai Medical Center.The U.S. Government may thus have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the signaling of estrogen and resistance tothe same. In particular embodiments, the invention relates to anintracellular estradiol binding protein, a gene encoding the same andvarious cell lines overexpressing the same.

BACKGROUND OF THE INVENTION

Estrogens are clinically important in both men and women. They affectgrowth, differentiation, and the development of reproductive tissues,and also play a role in a variety of diseases. For instance, estrogenmaintains bone density, and in the cardiovascular system, estrogenexerts anti-atherosclerotic effects by lowering circulating cholesterollevels.

Controlling the levels and/or effects of estrogen is important in mostforms of breast cancer. More than 1.2 million people will be diagnosedwith breast cancer this year worldwide. In the United States alone,nearly 211,300 women and 1,300 men are newly diagnosed with breastcancer each year. It is the second leading cause of cancer deaths inwomen today and is the most common cancer among women, excluding cancersof the skin.

Estrogen receptors are specialized proteins that bind to estrogen. Theseproteins are found in significant quantities within certainestrogen-sensitive tissues. Cells within breast tissue contain estrogenreceptors, for example, and the binding of estrogen to the estrogenreceptors stimulates these cells to proliferate. Many breast cancertumors also contain significant levels of estrogen receptors, and aretherefore called “estrogen receptor positive” (“ER+”).

One conventional method used to discontinue or slow down the growth andproliferation of breast cancer cells is to reduce the effects ofestrogen. The growth of ER+ breast cancer cells can generally becontrolled by blocking the estrogen receptors, lowering hormone levels,and/or reducing the number of receptors available to receive growthsignals. A conventional method to treat or help prevent the occurrenceof breast cancer is by administering selective estrogen receptormodulators (“SERMs”), which block the estrogen receptors by preventingthe growth signals from reaching the cells. SERMs target specificestrogen receptors in the body and either stimulate or depress anestrogen-like response, depending upon the particular organ. In breastcells, SERMs have antagonistic properties and block the effects ofestrogen, thereby slowing the growth of breast cancer cells.

Tamoxifen (available under the trade name NOLVADEX from AstraZenica PLC;London, UK) is a commonly used SERM, which is used to treat advanced andearly stage breast cancer. Tamoxifen is also used as therapy for theprimary prevention of breast cancer. Although tamoxifen has been used totreat breast cancer for nearly twenty years, it has some seriousdrawbacks. Tamoxifen therapy may increase the risk of cancer of theuterine lining (i.e., endometrial cancer and sarcoma), blood clotswithin deep veins (i.e., deep vein thrombosis), blood clots in the lungs(i.e., pulmonary embolism), and cataracts. Other adverse side effectscan include hot flashes, vaginal discharge, and menstrualirregularities.

Controlling the levels and/or effects of estrogen is also important inthe treatment and prevention of osteoporosis. Osteoporosis is a commonskeletal disorder characterized by a progressive decrease in bone massand density; causing bones to become abnormally thin, weakened, andeasily fractured. Although bone density naturally begins to decrease atapproximately 35 years of age, women are disproportionately at risk forosteoporosis after menopause due to declining production of estrogen.After menopause, in women who are not receiving hormonal therapy,estradiol levels are generally about 10-20 pg/ml. The average level ofestradiol needed to maintain healthy bones in menopausal women is about40-50 pg/ml. Osteoporosis is the most significant health hazardassociated with menopause; it affects 25% of women over the age of 65.

Osteoporosis is a significant public health threat for an estimated 44million Americans. In the United States today, 10 million individualsare estimated to already have the disease, and almost 34 million moreare estimated to have low bone mass, placing them at increased risk forosteoporosis. Of the 10 million Americans estimated to haveosteoporosis, 8 million are women and only 2 million are men.

Because estrogen is associated with the proliferation of cells, theclinical aim in osteoporosis treatment is to increase the effect ofestrogen (the opposite clinical goal of many breast cancer treatments).Conventional osteoporosis therapies include antiresorptive drugs,bone-building agents, and non-pharmacological intervention.Bisphosphonates are antiresporptive drugs that are widely used for theprevention and treatment of osteoporosis; they inhibit the breakdown andremoval of bone (i.e., resorption) and are typically the first choicefor osteoporosis treatment and prevention. However, in addition toadverse side effects, such as abdominal pain, nausea, and muscle andjoint pain, some patients who take bisphosphonates also develop severedigestive reactions including irritation, inflammation or ulceration ofthe esophagus. These reactions can cause chest pain, heartburn ordifficulty or pain upon swallowing. Raloxifene is another SERM commonlyused to treat osteoporosis, but it carries the risk of blood clots andmay cause a variety of side effects, including coughing blood, severeheadaches, loss of speech, coordination or vision, pain or numbness inthe arms, chest or legs, and shortness of breath. Still anotherconventional treatment for osteoporosis is estrogen-progestin therapy,but this approach is associated with side effects such as vaginalbleeding, bloating, nausea, headaches, and fluid retention.Estrogen-progestin therapy is no longer a first-line treatment forosteoporosis in postmenopausal women because of increases in the risk ofbreast cancer, stroke, blood clots, and perhaps coronary disease.

Conventional treatments for estrogen-related disease have substantialdrawbacks; many are only partially effective and have adverse sideeffects, and few provide a cure for associated conditions. Presentconventional methods to treat estrogen-related diseases may not besuitable for every patient. For the foregoing reasons and others, thereis a need for a clinical intervention that can be used to control theregulation of estrogen signaling. Such an intervention would be animportant tool to treat or prevent diseases and health conditions thatare related to levels of estrogen; for example, breast cancer andosteoporosis. An understanding of the biomolecular pathway responsiblefor these conditions would be of significant importance in treating, andultimately curing these conditions. A cell line that can be used as aclinical model in testing therapeutic interventions and diagnostictechniques would also be quite useful in this regard.

SUMMARY OF THE INVENTION

Various embodiments of the present invention provide a novelintracellular estradiol binding protein (“IEBP”), as well as thepolynucleotide that encodes it. While not wishing to be bound by anytheory, it is believed that the biological activity of estrogen may bemodulated by IEBP as, for example, by inhibiting or enhancing itsexpression or signaling. More particularly, it is believed that byincreasing the levels of IEBP, the signaling of estrogen is inhibited.Conversely, suppressing, inhibiting or otherwise lowering the levels ofIEBP enhances estrogen signaling. IEBP binds to 17β-estradiol (E₂), andinhibits estrogen response element transactivation by competing with theestrogen receptor (ER) to bind to E₂.

Further embodiments of the present invention describe cells and celllines that include the polynucleotide that encodes IEBP. Still furtherembodiments of the present invention describe cells and cell lines thatproduce and/or overexpress IEBP.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousfeatures of embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a comparative analysis of the homology between IEBP fromNew World primate (“NWP”) cells and human heat shock protein-27(“hsp27”) in accordance with an embodiment of the present invention.FIG. 1A depicts the full-length deduced amino acid sequence for IEBPcompared to human hsp27, and the human α-crystallins A (“hCrys A”) and B(“hCrys B”). The shaded areas depict regions of high sequence homologyamong all four molecules. The underlined regions denote areas ofsequence homology with the ATP- and substrate-binding domains of humanheat shock proteins in the −90 and −70 families. FIG. 1B depicts thepercentage homology values for an IEBP amino acid sequence compared tohsp27, hCrys A and hCrys B.

FIG. 2 depicts an analysis of estrogen response element (“ERE”)luciferase activity of IEBP and estrogen response element bindingprotein (“ERE-BP”) in accordance with an embodiment of the presentinvention. Expression constructs containing cDNA for the New Worldprimate ERE-BP and/or the New World primate IEBP were transientlyco-transfected with an estrogen-responsive luciferase reporter plasmidinto the ERα+ human MCF-7 breast cancer cell line, in the absence orpresence of 10 nM 17β-estradiol (E₂). Data are the mean of triplicatedeterminations of luciferase activity. *=statistically significantcompared to control transfectants, P<0.001. FIG. 2 suggests that IEBPcooperates with ERE-BP to squelch ERE-directed transactivation.

FIG. 3 illustrates estrogen's role in IEBP-mediated suppression ofERE-directed transactivation and depicts an analysis of cytoplasmicbinding, in accordance with an embodiment of the present invention. FIG.3A depicts an effect of IEBP on ERE promoter-reporter luciferaseactivity in wild-type 6299 breast cancer cells from an Old World primatehost stably transfected with vector alone (open bars) and threedifferent subclones of cells stably transfected with the full lengthIEBP cDNA, in the absence (left panel) or presence (right panel) of17β-estradiol (E₂ 10 nM; closed bars). Data are the mean of triplicatedeterminations of luciferase activity. *=statistically different fromvector alone transfectants, P<0.001. FIG. 3B depicts displacement of[³H]17β-estradiol (E₂) in postnuclear extracts of IEBP stablytransfected cell lines. Data are the mean of triplicate determinationsof % maximal [³H]17β-estradiol displaced by increasing doses of E₂(0.1-100 nM) in vector only controls and the three IEBP stabletransfectant cell lines. FIG. 3 suggests that IEBP-mediated suppressionof ERE-directed transactivation is associated with increased cytoplasmicbinding of estrogen.

FIG. 4 depicts electromobility shift assays (“EMSAs”) usingdouble-strand consensus ERE as probe, and recombinant human ERα and/orERE-affinity-purified ERE-BP as protein in accordance with an embodimentof the present invention. IEBP (lane 7, panel A; lanes 3 & 4, panel B)neither bound to ERE nor competed with the ER for binding to ERE (lane6, panel A; lanes 2 & 3, panel C) in the presence or absence of 100 nM17β-estradiol (E₂). The ER-ERE complex was supershifted by addinganti-hsp27 antibody with or without 100 nM E₂ (lanes 4 & 5; panel C).FIG. 4 suggests that the effects of IEBP on ERE-mediated transcriptionare not due to direct interaction with the ERE or disruption of ER-EREcomplex formation.

FIG. 5A depicts an EMSA using double-strand consensus ERE as probe andrecombinant human ERα, human hsp27 and anti-human hsp27 antibody ascomplexing protein in the presence or absence of 100 nM 17β-estradiol(E₂) in accordance with an embodiment of the present invention. FIG. 5Bdepicts immunoprecipitation of the protein constituents of postnuclearextracts of vector alone and IEBP-transfected Old World primate 6299breast cancer cells with anti-human hsp27 (left panel) and anti-humanERα (right panel) followed by detection with anti-human ERα antibody andanti-human hsp27 antibody, respectively. FIG. 5 suggests that there is adirect association between the human ERα and hsp27-like proteins.

FIG. 6 depicts 17β-estradiol-regulated expression of IEBP and itsinteraction with ERα in accordance with an embodiment of the presentinvention. FIG. 6A depicts a 17β-estradiol-mediated increase inexpression of hsp27 in wild-type breast cells. Shown is Western blotanalysis of hsp27 in wild-type breast cells in the absence or presenceof increasing doses of E₂ (0.1-100 nM). FIG. 6B depicts a yeasttwo-hybrid analysis of ligand 17β-estradiol (E₂)-dependent interactionof hsp27 with ERα. AH109 yeast cells were cotransfected with a Gal4 DNAbinding domain-ERα fusion protein plasmid, and colonies growth-selectedusing Leu-/Trp-/His-/Ade-medium containing either E₂ (10 nM), the ERantagonist tamoxifen (Tam, 10 nM) or vehicle only (none). FIG. 6Cdepicts a GST pull-down analysis of ER interacting proteins. Proteinextracts of cells overexpressing IEBP were incubated with either anER-GST or glucocorticoid receptor (GR)-GST fusion protein or GST proteinalone. GST-bound proteins were separated on SDS-PAGE and probed with ananti-hsp27 antibody. These results suggest that the ER-IEBP interactionis promoted by ligand 17β-estradiol but not the SERM tamoxifen.

FIG. 7 depicts normal and IEBP-mediated squelching of ER-ERE-directedtransactivation in accordance with an embodiment of the presentinvention. FIG. 7A depicts normal transcriptional events with17β-estradiol (E₂)-bound ER homodimer interacting with ERE to increasetranscription. FIG. 7B depicts “squelched” transcriptional events underthe influence of IEBP; the interposition of the E₂-binding IEBP betweenER and ligand leads to disruption of ER dimerization, the ER-EREinteraction and transactivation.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that methods to regulate estrogen signaling canbe derived from understanding hormone resistance in New World primates.Compared to Old World primates, including man, New World primatesdisplay relative resistance to adrenal, gonadal and vitamin Dsterol/steroid hormones, including 17β-estradiol. In female New Worldprimates, this hormone-resistant phenotype is characterized by elevatedconcentrations of plasma estradiol and progesterone. The precisemechanism for this hormone resistance in New World primates is not fullyunderstood, however, it does not involve aberrant expression of nuclearreceptors for specific hormones, which is the principal cause of hormoneresistance in humans. Instead, hormone resistance in New World primatecells appears to be due to epigenetic factors which result either inlow-affinity receptor-steroid binding kinetics or attenuation ofreceptor-DNA interaction. For instance, studies of glucocorticoidresistance in New World primate cells have shown increased expression ofthe heat-shock protein (hsp)90-associated FK 506-binding immunophilinFKBP51, which inhibited ligand binding to glucocorticoid receptors by74%.

Vitamin D resistance in New World primates appears to be due to aberrantexpression of hsp-70-like intracellular vitamin D binding proteins(“IDBPs”) and a dominant negative-acting vitamin D response elementbinding protein (“VDRE-BP”), the latter being homologous toheterogeneous nuclear ribonuclear protein A (“hnRNPA”). In a similarfashion to vitamin D, estrogen resistance in New World primates isassociated with the overexpression of two compensatory proteins: anintracellular estradiol binding protein (“IEBP”) and anon-receptor-related estrogen response element binding protein(“ERE-BP”).

By gaining an understanding of the biochemical mechanisms behindestrogen resistance and the high levels of circulating steroid hormonesin New World primates, new opportunities for treating and diagnosingdiseases related to estrogen and other steroidal hormones have beenachieved. The present invention is based on the surprising results ofthe inventors' research on estrogen resistance in New World primates. Asnoted above, they found that this resistance to estrogen is associatedwith the overexpression of two compensatory proteins: IEBP and ERE-BP.

Estrogen effects are generally mediated through the estrogen receptor(“ER”). Typically, the ER is activated when it binds to its ligandbinding domain (i.e., estrogen). The classical pathway for ER signalingis mediated by receptor binding to the estrogen response element(“ERE”), which is a specific DNA sequence to which the ER binds withhigh affinity. The ER undergoes a conformational change as a result ofligand binding, DNA binding, and phosphorylation by cell signalingpathways. This conformational change enables it to activatetranscription. If the ER ligand is inhibited or if there is not enoughestrogen for the ER dimer, then ER cannot be activated and thetranscription of ERE will decrease or not run.

IEBP is believed to be a member of the heat shock protein-27 (“hsp27”)family. Hsp27 was first identified in extracts of human breast cancercells as a heat shock and estrogen responsive protein; features that arecharacteristic of IEBP. A comparison of the homology of cDNA betweenhuman hsp27 and IEBP in New World primates suggests that there is 89.4%identity in 292 nt overlap.

As is described in further detail below, the term “IEBP” as used hereinrefers not only to proteins having the amino acid residue sequence ofnaturally occurring IEBPs (such as human hsp27 protein), but also refersto other equivalent proteins such as functional derivatives and variantsof the naturally occurring or synthetic IEBP, as well as compounds withactive sites that function in a manner similar to IEBP, whether thesecompounds are themselves naturally occurring or synthetic.

Like hsp27, IEBP expression was increased in response to heat shock.Furthermore, IEBP expression is more prominent in females than in maleswhile diminished in the female breast after ovariectomy. Thesecharacteristics attest to the estrogen responsiveness of IEBPexpression. Chen, et al., “Purification and Characterization of a NovelIntracellular 17β-Estradiol Binding Protein in Estrogen-Resistant NewWorld Primate Cells,” J. Clin. Endocrinol. Metab.,” 88: 501-504 (2003).While not wishing to be bound by any theory, it is believed that the hsp27-related IEBP binds to the ligand binding domain of estrogen receptoralpha (ERα) and acts to squelch 17β-estradiol (“E₂”)-ERα-mediatedtranscription. By binding to the ligand binding domain of ERα (i.e.,E₂), it is believed that IEBP inhibits ERE transactivation because itcompetes with ERα for E₂. IEBP competes with ERα for ligand binding,squelching 17β-estradiol-ER-directed signaling. It is further believedthat IEBP does not bind with ERE nor does it compete with ER to bind toERE to inhibit transactivation.

IEBP acts as an extra-nuclear depot for estrogen binding in a mannerthat is distinct from the ER to which it is linked. IEBP does not appearto directly influence ERα expression. The data from the inventors' studyindicate that the hsp 27-related IEBP acts as a corepressor or chaperonefor the cytoplasmic ER, with either dissociation or inactivation of thisfunction upon estrogen binding.

ERE-BP is a member of the hnRNP C (heterogeneous nuclearribonucleoprotein) family. Although the central RNP-containing domain ofERE-BP bears a high degree of sequence similarity to other hnRNP's, itis not clear whether these same RNA binding sites are also responsiblefor the binding of ERE-BP to DNA. Regardless of the primary structuralsimilarities between hnRNP's and ERE-BP, ERE differs from the classicalprofile of hnRNP in some aspects. For instance, ERE-BP is not confinedto the nuclear compartment of the cell. Based on recent studies, ERE-BPwas isolated in post-nuclear extracts of New World primate cells as wellas from both the cytoplasmic and nuclear compartments ofestrogen-resistant cells. Additionally, ERE-BP appears to be versatilein its ability to bind nucleic acid; ERE-BP can bind to single- ordouble-stranded DNA and may also interact with RNA. Chen et al.,“Cloning and Expression of a Novel Dominant-Negative-Acting EstrogenResponse Element-Binding Protein in the Heterogeneous NuclearRibonucleoprotein Family,” J Biol Chem, 273: 31352-31357 (1998).

It is believed that ERE-BP acts to squelch ER-ERE transactivation bycompeting with ER to bind to ERE. ERE-BP binds to ERE, and not theligand of E₂. ERE-BP acts in a dominant negative cis-acting mode tosquelch transactivation by competing with ER for its response element.By acting directly with ERE and interfering with ER binding, ERE-BPsilences ER action.

IEBP can cooperate with ERE-BP by acting as an intracellular repositoryfor E₂ or by binding to the ligand-binding domain of ER. In either case,the net effect is that IEBP interrupts ER-ER homodimerization, therebylegislating estrogen resistance. Based on the amino acid sequence oftryptic fragments of a protein purified by E₂ affinity chromatographyfrom post-nuclear extracts of estrogen-resistant NWP cells, theinventors were able to design degenerate primers that enabledamplification of an IEBP cDNA with similarity to the “small heat shockproteins,” including human hsp27 and crystallins A and B (FIG. 1A). Thehigh degree of sequence identity, 87% (FIG. 1B), between the New Worldprimate IEBP and the human hsp27 indicates that IEBP is likely theinterspecies homology of human hsp27. The family of small heat shockproteins (sHsps; 15-42 kDa), which includes hsp27, is encountered inboth pro- and eukaryotes. In the human genome, hsp27 is encoded by fourdifferent genes on chromosomes 3, 7, 9 and X; such redundancy indicatesthat the encoded protein(s) is critical for survival of the host. Liketheir counterparts in the 70 kDa, 90 kDa and 60 kDa molecular weightrange, the sHsps (1) are upregulated by cell “stress”; including heatstress which is transduced by heat shock factors interacting withspecific heat shock enhancer elements in the promoter of hsp genes, and(2) can function as “molecular chaperones” protecting the structural andfunctional integrity of the intracellular proteins to which they arebound. Unlike heat shock proteins in the 70, 90 and 60 kDa families,sHsps appear to: (1) be less homologous in their amino acid sequence,(2) play a central role in preventing apoptosis, (3) be crucial for theorganization of the cytoskeleton and microfilamental structures thereinand (4) be needed for self-oligimerization, providing for the refractorynature of the human lens.

Although the N- and C-terminal amino acid sequences may varyconsiderably among sHsp family members and between species, the generaldomain structure of the sHsp molecules remains highly conserved throughevolution. A central α-crystallin domain of ˜90 residues is bounded bythe variable N-terminal and C-terminal extensions. The C-terminalextension, a polar structure, is now considered to be the prime mediatorof the molecules' chaperone function, while the conserved α-crystallinand variable N-terminal domains are thought to be essential formultimerization of the sHsps. In addition to being highly homologouswith human hsp27, when compared to the α-crystallins (FIGS. 1A and 1B)the IEBP isolated from estrogen-resistant New World primate cells herewas determined to possess the typical conserved central α-crystallincore domain flanked by variable N- and C-terminal extensions.Considering that IEBP was isolated by its ability to adhere to anE₂-affinity support, the IEBP sequence was scanned for the presence ofan estrogen binding site. As shown in FIG. 1A, a sequence with 38%identity to a 21 amino acid stretch of the ligand binding domain of theERα was detected in the highly conserved α-crystallin core domain of themolecule. Although formal mapping studies were not completed, it ispresumed that it is this part of the IEBP, and its human homology hsp27,that is responsible for the enhancement of E₂ binding in cellsconstitutively overexpressing IEBP (FIG. 3B). Moreover, because thisputative E₂ binding subdomain resides in the conserved α-crystallindomain of the sHsps, it is possible that estrogen binding may be afunction of other members of the sHsp27 family. Although there residessome sequence identity to the ATP-binding/ATPase domain prototypical ofthe larger heat shock proteins (e.g., hsp70, hsp90 and hsp60) in theconserved α-crystallin domain, the role, if any, ATP has in governingthe function of IEBP and other hsp70-like proteins remains to bedetermined.

In addition to bearing the above-mentioned enhancer cis elements thatcan interact with heat shock factors, the hsp 27 promoter also containsan estrogen response element (ERE) half site in direct proximity to anSp1 site and the TATA box. While this ERE half site can be shown tointeract with the ERα, E₂-directed transactivation of the hsp27 gene, asreported by a number of laboratories, does not require the EREhalf-site. The inventors' studies confirm that the anti-humanhsp27-reactive IEBP is an estrogen-responsive gene product, beingmarkedly up-regulated after overnight exposure to ER-saturatingconcentrations of E₂ (FIG. 5A). So IEBP, and presumably its humanhomology hsp27, is an estrogen up-regulated gene product that can, inturn, bind the same hormone. These results indicate that E₂ canup-regulate expression of an E₂-interacting protein that, in turn, cansquelch E₂-ERα-ERE-directed transactivation (FIGS. 2 and 3). Thissuggests that transcriptional down-regulation of IEBP or hsp27 geneexpression might be achieved by the product of that gene through itsability to squelch, but not completely subdue (FIG. 2, bars 3 and 4)estrogen-driven expression. In other words, it is possible that hsp27 orIEBP could be auto-regulated (i.e., when E₂-ERα-directed hsp27expression goes up, it produces a protein(s) that dampens subsequentE₂-ER enhancer action at the level of the hsp27-promoter). Such anegative feedback system would serve to reostatically regulateE₂-promoted transcactivation.

Human breast cancers that harbor the ERα are susceptible toestrogen-directed growth advantage. This has led to the broad usage ofSERMs as adjuvant chemotherapeutic agents in this disease. Themechanism(s) by which occupancy of the ERα by E₂ affects this change intumor cell growth and proliferation remain an area of intenseinvestigation. One of the genes that is activated in humanERα-expressing breast cancer cells by E₂ exposure is hsp27, the humanhomology of the New World primate IEBP reported here; similarly,estrogen-driven hsp27 expression can be squelched by exposure of cellsto SERMs. This has led to the investigation of hsp27, like ERα, as ahuman breast cancer tumor marker with hsp27 tumor expression currentlysuggested to be a “downstream” indicator of estrogen-ERα interaction intumor cells. In some, but not all studies, hsp27 expression has beenshown to correlate with ERα expression. Therefore, it is of note that inthe co-immunoprecipitation, yeast two-hybrid and GST pull-down assayscarried out as part of the current studies (FIGS. 5B and 6) there wasevidence for a direct protein-protein interaction between the hsp27-likeIEBP and ERα that was promoted by the presence of E₂ and hindered byexposure to the clinically-useful SERM tamoxifen. These data suggestthat co-expression of the ERα and IEBP by breast cancer cells may befunctionally as well as temporally linked to one another. Theconsequences of the additive, dominant-negative-acting squelching ofE₂-ERα-ERE-directed transcription of IEBP (hsp27) and the hnRNP-relatedERE-BP on breast cancer cell behavior in vitro are currently underinvestigation.

The present invention thus relates to a novel IEBP and a polynucleotidethat encodes the same; particularly, isolated and/or purified IEBP andits corresponding coding sequence. Further embodiments of the presentinvention relate to cells and cell lines that include the polynucleotidethat encodes IEBP, as well as cells and cell lines that produce and/oroverexpress IEBP. The inventive IEBP deduced peptide is illustrated asSEQ ID NO:1. The polynucleotide sequence that encodes this IEBP peptideis illustrated as SEQ ID NO:2. The polynucleotide sequence correspondingto a full-length cDNA for IEBP used in various embodiments of thepresent invention is illustrated as SEQ ID NO:3. This cDNA was clonedfrom the estrogen-resistant cell line B95-8, as described in greaterdetail in the ensuing Examples.

Use of the terms “isolated” and/or “purified” in the presentspecification and claims as a modifier of DNA, RNA, polypeptide orproteins means that the DNA, RNA, polypeptide or proteins so designatedhave been produced in such form by the hand of man, and thus areseparated from their native in vivo cellular environment. As a result ofthis human intervention, the recombinant DNAs, RNAs, polypeptide andproteins of the invention are useful in ways described herein that theDNAs, RNAs, polypeptide or proteins as they naturally occur are not.

Presently preferred IEBP proteins of the invention include amino acidsequences that are substantially the same as the amino acid sequence SEQID NO:1 and fragments thereof, as well as biologically active, modifiedforms thereof. Those of skill in the art will recognize that numerousresidues of the above-described sequences can be substituted with otherchemically, sterically and/or electronically similar residues withoutsubstantially altering the biological activity of the resulting receptorspecies. In addition, larger polypeptide sequences containingsubstantially the same sequence as SEQ ID NO:1 therein (e.g., splicevariants) are contemplated.

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70%identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the protein defined by the reference amino acid sequence. Preferably,proteins having “substantially the same amino acid sequence” will haveat least about 80%, more preferably 90% amino acid identity with respectto the reference amino acid sequence; with greater than about 95% aminoacid sequence identity being especially preferred. It is recognized,however, that polypeptide (or nucleic acids referred to hereinbefore)containing less than the described levels of sequence identity arisingas splice variants or that are modified by conservative amino acidsubstitutions or by substitution of degenerate codons are alsoencompassed within the scope of the present invention.

The terms “biologically active” or “functional,” when used herein as amodifier of the IEBP protein of this invention or polypeptide fragmentthereof, refers to a polypeptide that exhibits at least one of thefunctional characteristics attributed to IEBP. For example, onebiological activity of IEBP is the ability to impart estrogen resistanceto mammalian cells when overexpressed therein.

The IEBP proteins of the invention may be isolated by methods well knownin the art; for instance, by various recombinant expression systems,precipitation, gel filtration, ion-exchange, reverse-phase and affinitychromatography and the like. Other well-known methods are described, forexample, in Deutscher et al., Guide to Protein Purification: Methods inEnzymology Vol. 182, (Academic Press, (1990)), which is incorporatedherein by reference. Alternatively, the isolated polypeptide of thepresent invention can be obtained using well-known recombinant methodsas described, for example, in Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press (1989).

An example of a means for preparing the invention polypeptide(s) is toexpress nucleic acids encoding IEBP in a suitable host cell, such as abacterial cell, a yeast cell, an amphibian cell (e.g., oocyte), or amammalian cell using methods well known in the art, and recovering theexpressed polypeptide, again using well known methods. The IEBPpolypeptide of the invention may be isolated directly from cells thathave been transformed with expression vectors as described herein. Theinvention polypeptide, biologically active fragments and functionalequivalents thereof can also be produced by chemical synthesis. Forexample, synthetic polypeptide can be produced using Applied Biosystems,Inc. Model 430A or 431A automatic peptide synthesizer (Foster City,Calif.) employing the chemistry provided by the manufacturer.

Also encompassed by the term IEBP are polypeptide fragments orpolypeptide analogs thereof. The term “polypeptide analog” includes anypolypeptide having an amino acid residue sequence substantiallyidentical to a sequence specifically shown herein (i.e., SEQ ID NO:1) inwhich one or more residues have been conservatively substituted with afunctionally similar residue and which displays the ability to mimicIEBP as described herein. Examples of conservative substitutions includethe substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another. The phrase“conservative substitution” also includes the use of a chemicallyderivatized residue in place of a non-derivatized residue, provided thatsuch polypeptide displays the requisite binding activity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include, for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-imbenzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For example, 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions of residues, relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

The present invention also provides compositions containing anacceptable carrier and any of an isolated, purified IEBP polypeptide, anactive fragment or polypeptide analog thereof, or a purified, matureprotein and active fragments thereof, alone or in combination with oneanother. These polypeptides or proteins can be recombinantly derived,chemically synthesized or purified from native sources. As used herein,the term “acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as phosphate buffered saline (“PBS”)solution, water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents.

In accordance with another embodiment of the present invention, thereare provided isolated nucleic acids, which encode the IEBP proteins ofthe invention, and fragments thereof. The nucleic acid moleculesdescribed herein are useful for producing invention proteins when suchnucleic acids are incorporated into a variety of protein expressionsystems known to those of skill in the art. In addition, such nucleicacid molecules or fragments thereof can be labeled with a readilydetectable substituent and used as hybridization probes for assaying forthe presence and/or amount of an IEBP gene or mRNA transcript in a givensample. The nucleic acid molecules described herein and fragmentsthereof are also useful as primers and/or templates in a PCR reactionfor amplifying genes encoding the invention protein described herein.

The term “nucleic acid” (also referred to as polynucleotides)encompasses ribonucleic acid (“RNA”) or deoxyribonucleic acid (“DNA”),probes, oligonucleotides and primers. DNA can be either complementaryDNA (“cDNA”) or genomic DNA (e.g., a gene encoding an IEBP protein). Onemeans of isolating a nucleic acid encoding an IEBP polypeptide is toprobe a mammalian genomic library with a natural or artificiallydesigned DNA probe using methods well known in the art. DNA probesderived from the IEBP gene are particularly useful for this purpose. DNAand cDNA molecules that encode IEBP polypeptide can be used to obtaincomplementary genomic DNA, cDNA or RNA from mammalian (e.g., Old Worldprimate, New World primate, human, mouse, rat, rabbit, pig and the like)or other animal sources, or to isolate related cDNA or genomic clones bythe screening of cDNA or genomic libraries by conventional methods.Examples of nucleic acids are RNA, cDNA, or isolated genomic DNAencoding an IEBP polypeptide. Such nucleic acids may include, but arenot limited to, nucleic acids comprising SEQ ID NO:2, SEQ ID NO:3,alleles thereof, or splice variant cDNA sequences thereof.

As used herein, the phrases “splice variant” or “alternatively spliced,”when used to describe a particular nucleotide sequence encoding aninvention polypeptide, refers to a cDNA sequence that results from thewell known eukaryotic RNA splicing process. The RNA splicing processinvolves the removal of introns and the joining of exons from eukaryoticprimary RNA transcripts to create mature RNA molecules of the cytoplasm.Methods of isolating splice variant nucleotide sequences are well knownin the art. For example, one of skill in the art may employ nucleotideprobes derived from the IEBP encoding DNA of SEQ ID NO:2, SEQ. ID NO:3,alleles thereof, splice variants thereof or fragments thereof about 10to 150 nucleotides long and their antisense nucleic acids to screen thecDNA or genomic library of the same or other species as describedherein.

In one embodiment of the present invention, DNAs encoding the IEBPprotein of this invention comprise SEQ. ID NO:2, SEQ. ID NO:3, allelesthereof, splice variants thereof and fragments thereof and antisensenucleic acids thereof.

As employed herein, the term “substantially the same nucleotidesequence” refers to DNA having sufficient identity to the referencepolynucleotide such that it will hybridize to the reference nucleotideunder moderately stringent hybridization conditions. In one embodiment,DNA having substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in SEQ ID NO:1, or a larger amino acid sequenceincluding SEQ ID NO:1. In another embodiment, DNA having “substantiallythe same nucleotide sequence” as the reference nucleotide sequence hasat least 60% identity with respect to the reference nucleotide sequence.DNA having at least 70%, more preferably at least 90%, yet morepreferably at least 95% identity to the reference nucleotide sequence ispreferred.

The present invention also encompasses nucleic acids which differ fromthe nucleic acids shown in SEQ ID NO:2 and SEQ ID NO:3, but which havethe same phenotype. Phenotypically similar nucleic acids are alsoreferred to as “functionally equivalent nucleic acids.” As used herein,the phrase “functionally equivalent nucleic acids” encompasses nucleicacids characterized by slight and non-consequential sequence variationsthat will function in substantially the same manner to produce the sameprotein product(s) as the nucleic acids disclosed herein. In particular,functionally equivalent nucleic acids encode polypeptides that are thesame as those disclosed herein or that have conservative amino acidvariations, or that encode larger polypeptides that include SEQ ID NO:1.For example, conservative variations include substitution of a non-polarresidue with another non-polar residue, or substitution of a chargedresidue with a similarly charged residue. These variations include thoserecognized by skilled artisans as those that do not substantially alterthe tertiary structure of the protein.

Further provided are nucleic acids encoding IEBP polypeptides that, byvirtue of the degeneracy of the genetic code, do not necessarilyhybridize to the invention nucleic acids under specified hybridizationconditions. Preferred nucleic acids encoding the IEBP polypeptide of theinvention comprise nucleotides encoding SEQ ID NO:1 and fragmentsthereof. Exemplary nucleic acids encoding an IEBP protein of theinvention may be selected from the following:

-   -   (a) DNA encoding the amino acid sequence set forth in SEQ ID        NO:1,    -   (b) DNA that hybridizes to the DNA of (a) under moderately        stringent conditions, wherein said DNA encodes biologically        active IEBP, and    -   (c) DNA degenerate with respect to either (a) or (b) above,        wherein said DNA encodes biologically active IEBP.

As used herein, the term “degenerate” refers to codons that differ in atleast one nucleotide from a reference nucleic acid (e.g., SEQ ID NO:2 orSEQ ID NO:3), but encode the same amino acids as the reference nucleicacid. For example, codons specified by the triplets “UCU,” “UCC,” “UCA”and “UCG” are degenerate with respect to each other since all four ofthese codons encode the amino acid serine.

Hybridization refers to the binding of complementary strands of nucleicacid (i.e., sense:antisense strands or probe:target-DNA) to each otherthrough hydrogen bonds; similar to the bonds that naturally occur inchromosomal DNA. Stringency levels used to hybridize a given probe withtarget-DNA can be readily varied by those of skill in the art.

The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As is knownto those of skill in the art, the stability of hybrids is reflected inthe melting temperature (T_(m)) of the hybrids. In general, thestability of a hybrid is a function of sodium ion concentration andtemperature. Typically, the hybridization reaction is performed underconditions of lower stringency, followed by washes of varying, buthigher stringency. Reference to hybridization stringency relates to suchwashing conditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-DNA to bind a complementary nucleicacid that has about 60% identity, preferably about 75% identity, morepreferably about 85% identity to the target DNA; with greater than about90% identity to target-DNA being especially preferred. Preferably,moderately stringent conditions are conditions equivalent tohybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDSat 42° C., followed by washing in 0.2×SSPE, 0.2% SDS at 65° C.

The phrase “high stringency hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stablein 0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein). High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C.

The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5× Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS at50° C. Denhart's solution and SSPE are well known to those of skill inthe art as are other suitable hybridization buffers. See, e.g., Sambrooket al., supra.

Preferred nucleic acids encoding the invention polypeptide(s) hybridizeunder moderately stringent, preferably high stringency conditions tosubstantially the entire sequence or substantial portions (i.e.,typically at least 15-30 nucleotides of SEQ ID NO:2 or SEQ ID NO:3,although longer fragments are also contemplated as being within thescope of the present invention in this regard).

Site-directed mutagenesis of any region of IEBP cDNA is contemplatedherein for the production of mutant IEBP cDNAs. For example, theTransformer Mutagenesis Kit (available from Clontech) can be used toconstruct a variety of mis-sense and/or nonsense mutations to IEBP cDNA.

The inventive nucleic acids can be produced by a variety of methods wellknown in the art (e.g., the methods described herein, employing PCRamplification using oligonucleotide primers from various regions of SEQID NO:2, and the like).

In accordance with a further embodiment of the present invention,optionally labeled IEBP-encoding cDNAs or fragments thereof can beemployed to probe a library(ies) (e.g., cDNA, genomic and the like) foradditional nucleic acid sequences encoding related novel mammalian IEBPproteins. Construction of mammalian cDNA and genomic libraries,preferably a human library, is well-known in the art. Screening of sucha cDNA or genomic library is initially carried out under low-stringencyconditions, which comprise a temperature of less than about 42° C., aformamide concentration of less than about 50% and a moderate to lowsalt concentration.

Presently preferred probe-based screening conditions comprise atemperature of about 37° C., a formamide concentration of about 20% anda salt concentration of about 5× standard saline citrate (SSC; 20×SSCcontains 3M sodium chloride, 0.3M sodium citrate; pH 7.0). Suchconditions will allow the identification of sequences which have asubstantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Preferably,hybridization conditions will be selected that allow the identificationof sequences having at least 70% homology with the probe, whilediscriminating against sequences which have a lower degree of homologywith the probe. As a result, nucleic acids having substantially the same(i.e., similar) sequences as the coding region of the nucleic acids ofthe invention are obtained.

As used herein, a nucleic acid “probe” is single-stranded DNA or RNA oranalogs thereof that has a sequence of nucleotides that includes atleast 14, preferably at least 20, more preferably at least 50,contiguous bases that are the same as (or the complement of) any 14 ormore contiguous bases set forth in any of SEQ ID NO:2 or SEQ ID NO:3.Preferred regions from which to construct probes include 5′ and/or 3′coding regions of SEQ ID NO:2. In addition, the entire cDNA encodingregion of an invention IEBP protein, or the entire sequencecorresponding to SEQ ID NO:2 or SEQ ID NO:3, may be used as a probe.Probes may be labeled by methods well-known in the art, as describedhereinafter, and used in various diagnostic kits.

In accordance with yet another embodiment of the present invention,there is provided a method for the recombinant production of the IEBPprotein of the invention by expressing the above-described nucleic acidsequences in suitable host cells. Any cell or cell line may be used as ahost cell in accordance with alternate embodiments of the presentinvention to create a cell or cell line that produces or overexpressesIEBP. The modified cells or cell lines generated from the host cells maybe, for example, estrogen-responsive (e.g., Old World primate 6299breast cells) or estrogen-resistant (e.g., B95-8 cells). Such cells andcell lines may be used, for example, in the screening of pharmaceuticalpreparations or to generate significant quantities of IEBP for inclusionin various inventive compositions. Other uses for these cells and celllines will be readily apparent to those of skill in the art. RecombinantDNA expression systems that are suitable to produce IEBP proteinsdescribed herein are also well known in the art. For example, theabove-described nucleotide sequences can be incorporated into vectorsfor further manipulation. As used herein, “vector” (or “plasmid”) refersto discrete elements that are used to introduce heterologous DNA intocells for either expression or replication thereof.

Suitable expression vectors are well-known in the art, and includevectors capable of expressing DNA operatively linked to a regulatorysequence, such as a promoter region that is capable of regulatingexpression of such DNA. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. In addition, vectors may containappropriate packaging signals that enable the vector to be packaged by anumber of viral virions (e.g., retroviruses, herpes viruses,adenoviruses) resulting in the formation of a “viral vector.”

As used herein, a “promoter region” refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis-acting or may be responsive totrans-acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (“CMV”) promoter, the mousemammary tumor virus (“MMTV”) steroid-inducible promoter, Moloney murineleukemia virus (“MMLV”) promoter and the like.

As used herein, the term “operatively linked” refers to the functionalrelationship of DNA with regulatory and effector nucleotide sequences,such as promoters, enhancers, transcriptional and translational stopsites, and other signal sequences. For example, operative linkage of DNAto a promoter refers to the physical and functional relationship betweenthe DNA and the promoter such that the transcription of such DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA.

As used herein, “expression” refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptide or proteins. If the polynucleic acid is derived from genomicDNA, expression may, if an appropriate eukaryotic host cell or organismis selected, include splicing of the mRNA.

Prokaryotic transformation vectors are well-known in the art and includepBlueskript and phage Lambda ZAP vectors (available from Stratagene; LaJolla, Calif.) and the like. Other suitable vectors and promoters aredescribed in detail in U.S. Pat. No. 4,798,885, the disclosure of whichis incorporated herein by reference in its entirety.

Other suitable vectors for transformation of E. coli cells include thepET expression vectors (available from Novagen; see U.S. Pat. No.4,952,496); for example, pET11a, which contains the T7 promoter, T7terminator, the inducible E. coli lac operator and the lac repressorgene, and pET 12a-c, which contain the T7 promoter, T7 terminator andthe E. coli ompT secretion signal. Another suitable vector is thepIN-IIIompA2 (see Duffaud et al., Meth. in Enzymology, 153:492-507,1987), which contains the Ipp promoter, the lacUV5 promoter operator,the ompA secretion signal and the lac repressor gene.

Exemplary eukaryotic transformation vectors include the cloned bovinepapilloma virus genome, the cloned genomes of the murine retrovirusesand eukaryotic cassettes such as the pSV-2 gpt system (described byMulligan and Berg, 1979, Nature, vol. 277:108-114), the Okayama-Bergcloning system (Mol. Cell Biol. Vol. 2:161-170, 1982) and the expressioncloning vector described by Genetics Institute (Science, vol.228:810-815, 1985). Each is available and provides substantial assuranceof at least some expression of the protein of interest in thetransformed eukaryotic cell line.

Particularly preferred base vectors that contain regulatory elementsthat can be linked to the invention IEBP-encoding DNAs for transfectionof mammalian cells are CMV promoter-based vectors, such as pcDNA1(available from Invitrogen; San Diego, Calif.); MMTV promoter-basedvectors, such as pMAMNeo (available from Clontech) and pMSG (availablefrom Pharmacia; Piscataway, N.J.); and SV40 promoter-based vectors, suchas pSVβ (available from Clontech).

In accordance with another embodiment of the present invention, thereare provided “recombinant cells” containing the nucleic acid molecules(i.e., DNA or mRNA) of the present invention. Methods of transformingsuitable host cells, preferably bacterial cells, and more preferably E.coli cells, as well as methods applicable for culturing said cellscontaining a gene encoding a heterologous protein, are generally knownin the art. See, e.g., Sambrook et al., supra.

Exemplary methods of introducing (transducing) expression vectorscontaining invention nucleic acids into host cells to produce transducedrecombinant cells (i.e., cells containing recombinant heterologousnucleic acid) are well-known in the art (see, e.g., Friedmann, 1989,Science, 244:1275-1281; Mulligan, 1993, Science, 260:926-932, each ofwhich is incorporated herein by reference in its entirety). Exemplarymethods of transduction include, for instance, infection employing viralvectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764), calciumphosphate transfection (see, e.g., U.S. Pat. Nos. 4,399,216 and4,634,665), dextran sulfate transfection, electroporation, lipofection(see, e.g., U.S. Pat. Nos. 4,394,448 and 4,619,794), cytofection,particle bead bombardment and the like. The heterologous nucleic acidcan optionally include sequences that allow for its extrachromosomal(i.e., episomal) maintenance, or the heterologous DNA can be caused tointegrate into the genome of the host (as an alternative means to ensurestable maintenance in the host).

Host organisms contemplated for use in the practice of the presentinvention include those organisms in which recombinant production ofheterologous proteins has been carried out. Examples of such hostorganisms include bacteria (e.g., E. coli), yeast (e.g., Saccharomycescerevisiae, Candida tropicalis, Hansenula polymorpha and P. pastoris;see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555 and4,855,231), mammalian cells (e.g., B95-8, Old World primate 6299,HEK293, CHO and Ltk cells), insect cells and the like. Presentlypreferred host organisms are bacteria. The most preferred bacteria is E.coli.

In one embodiment, nucleic acids encoding the IEBP proteins of theinvention may be delivered into mammalian cells, either in vivo or invitro using suitable viral vectors well-known in the art, e.g.,retroviral vectors, adenovirus vectors and the like.

Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing IEBP nucleic acidencoding an IEBP protein into mammalian cells are well known in the art.These viral vectors include, for example, Herpes simplex virus vectors(e.g., Geller et al., 1988, Science, 241:1667-1669), Vaccinia virusvectors (e.g., Piccini et al., 1987, Meth. in Enzymology, 153:545-563);CMV vectors (Mocarski et al., Viral Vectors; Y. Gluzman and S. H.Hughes, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1988, pp. 78-84), MMLV vectors (Danos et al., 1980, Proc. Nat'l. Acad.Sci. USA, 85:6469), adenovirus vectors (e.g., Logan et al., 1984, Proc.Nat'l. Acad. Sci. USA, 81:3655-3659; Jones et al., 1979, Cell,17:683-689; Berkner, 1988, Biotechniques, 6:616-626; Cotten et al.,1992, Proc. Nat'l. Acad. Sci. USA, 89:6094-6098; Graham et al., 1991,Meth. Mol. Biol., 7:109-127), adeno-associated virus vectors, retrovirusvectors and the like. See, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764.Especially preferred viral vectors are the adenovirus and retroviralvectors.

As used herein, “retroviral vector” refers to the well-known genetransfer plasmids that have an expression cassette encoding anheterologous gene residing between two retroviral LTRs. Retroviralvectors typically contain appropriate packaging signals that enable theretroviral vector, or RNA transcribed using the retroviral vector as atemplate, to be packaged into a viral virion in an appropriate packagingcell line (see, e.g., U.S. Pat. No. 4,650,764). Suitable retroviralvectors for use herein are described, for example, in U.S. Pat. No.5,252,479, and in WIPO publications WO 92/07573, WO 90/06997, WO89/05345, WO 92/05266 and WO 92/14829, incorporated herein by reference,which provide a description of methods for efficiently introducingnucleic acids into human cells using such retroviral vectors. Otherretroviral vectors include, for example, the MMTV vectors (e.g.,Shackleford et al., 1988, Proc. Nat'l. Acad. Sci. USA, 85:9655-9659) andthe like.

EXAMPLES

The following examples illustrate the biological activity of IEBP, aswell as methods for preparing cells and cell lines that produce and/oroverexpress IEBP. In the following examples, where indicated,experimental means were compared statistically using an unpairedStudent's t-test.

Example 1 Cell Culture

All cell lines were obtained from American Type Culture Collection(ATCC; Rockville, Md.). The estrogen-resistant NWP cell line B95-8,derived from the hormone resistant common marmoset (Callithrix jacchus),was maintained in RPMI-1640 medium. The estrogen-responsive OWP breastcell line 6299, derived from a rhesus monkey (Macaca mulatta), wasmaintained in DMEM (Irvine Scientific Irvine; Calif.). All cultures wereroutinely supplemented with 10% fetal calf serum (FCSI, GeminiBioproducts; Calabasas, Calif.), 100 units/ml penicillin, 100 μg/mlstreptomycin, 2 mM L-glutamine (both from GIBCO-BRL; Grand Island, N.Y.)and in atmosphere of 95% air, 5% CO₂. In some experiments, confluentcultures were preincubated for up to 48 hours in medium containing E₂(10 nM) prior to harvest and preparation of extracts.

Example 2 Preparation of Cellular Extracts

Postnuclear extracts of each cell line were prepared as described inChen et al., “Vitamin D and Gonadal Steroid-Resistant New World PrimateCells Express an Intracellular Protein Which Competes with the EstrogenReceptor for Binding to Estrogen Response Element,” J Clin Invest, 99:669-675 (1997). Harvested cells were washed twice in ice-coldphosphate-buffered saline (PBS) and twice washed with ETD buffer (1 mMEDTA, 10 mM Tris-HCL, 5 mM dithiothreitol (pH 7.4)) containing 1 mMphenylmethylsulfonylflouride (PMSF). The cell pellets were thenresuspended in ETD buffer and homogenized on ice in five 10-secondbursts. Nuclei, with associated nuclear steroid receptor proteins, werepelleted at 10,000×g for 30 min at 4° C.

Example 3 Molecular Cloning of the IEBP by Rapid Amplification ofComplementary Ends (RACE)

In previous studies, the inventors identified and characterized trypticfragments of an E₂-affinity column purified protein which correspondedto the putative IEBP in NWP B95-8 cells. Chen, et al., “Purification andCharacterization of a Novel Intracellular 17β-Estradiol Binding Proteinin Estrogen-Resistant New World Primate Cells,” J. Clin. Endocrinol.Metab., 88: 501-504 (2003). To clone the mRNA corresponding to thisprotein they used degenerate oligonucleotides corresponding to the aminoacid sequence of tryptic peptides to carry out RACE generation ofcandidate cDNA. Sequence analysis of the resulting deduced amino acidsequence for IEBP (FIG. 1A) revealed 87% sequence identity to the humanhsp27, 40% sequence identity to human α-crystallin chain A and 44%sequence identity to chain B (FIG. 1B). The amino-acid sequence alsoexhibited 28-38% identity with a 21 amino-acid overlap in theligand-binding domain of ERα, but there was no LXXLL motif typical ofthe steroid hormone receptor (data not shown).

Based on the amino acid of the N-terminal tryptic peptide (RVPFSL) ofIEBP, the inventors designed an IEBP-specific sense oligonucloetideprimer and its reversion antisense primer for 5′ and 3′-RACE. Poly (A)+RNA (2.5 μg) from B95-8 cells was used as the template to generate the5′- and 3′-ends of the IEBP cDNA with a BD MARATHON cDNA amplificationkit (Clontech Laboratories Inc.; Palo Alto, Calif.). Second-strand cDNAsynthesis and adapter ligation were performed as instructed in theenclosed manual. The adapter-ligated cDNA was then used as a templatefor annealing adapter- and IEBP-specific primers for the RACE reaction:5′-CGCAGGAGCGAGAAGGGGACGCG-3′ (SEQ ID NO:4) and5′-CGCGTCCCCTTCTCGCTCCTGC-3′ (SEQ ID NO:5) for the 5′- and 3′-RACE ofIEBP, respectively. A cDNA for the IEBP was generated by end-to-endamplification using specific 5′ and 3′ primers. The amplified productswere then subcloned into the pcDNA 3.1/V5/His/TOPO expression vector andsequenced by the Cedars-Sinai Medical Center Sequencing Core Facilityusing dye terminator cycle sequence reactions and ABI automatedsequencers.

Example 4 Transient Transfections

5×10⁵ estrogen-resistant NWP B95-8 or estrogen-responsive OWP breast6299 cells were seeded into 6-well plates in phenol red-free mediumcontaining 10% charcoal-stripped fetal calf serum (“FCS”) and allowed toproliferate to 80-90% confluence. Transfections were performed intriplicate with the combinations of DNA preparations set forth in Table1 to a maximum final concentration of 20 μg DNA/ml in LIPOTAXI solution(Stratagene; La Jolla, Calif.).

TABLE 1 DNA Preparations Used to Perform Transfections i. 5.5 μgERE-luciferase reporter plasmid ii. 0.5 μg ERα expression plasmid(pRShER) iii. 5.0 μg of IEBP or ERE-BP plasmid (in cDNA3.1his/v5 TOPOvector) iv. 5.0 μg β-galactosidase expression construct as internalcontrol v. pGEM-3z vector DNA as carrier (Promega, Madison, MI).

An equal volume of 20% FCS-supplemented, antibiotic-free medium wasadded to each well 5 hours after transfection followed by the additionof 10 nM E₂. After an additional 48 hours at 37° C., the cells werelysed, and luciferase and β-galactosidase activities were measured (FIG.2).

Example 5 Generation of Cell Lines Overexpressing IEBP

E₂-responsive OWP breast cells from cell line 6299 were incubated with5.0 ug pcDNA3.1/v5-His-TOPO IEBP plasmid in LIPOTAXI solution for 5hours followed by the addition of equal volume of 20% FCS-supplementedmedium. After incubation overnight, cells were split (1:10 ratio) andincubated with fresh medium containing 500 ug/ml of thegeneticin-selective antibiotic G418 sulfate (Life Technology; GrandIsland, N.Y.). This medium was replaced every 3-4 days, until stablecolonies formed. Single colonies were picked, transferred into a newdish, and incubated with medium containing selection antibiotic G418until confluence for further study.

Example 6 Ligand Binding Analysis

Specific [³H]17β-estradiol (“[³H]E₂”) binding was measured inpostnuclear extracts of vector-alone and the three IEBP stablytransfected cell lines (FIG. 3B). Briefly, postnuclear extracts isolatedas described above were reconstituted in NaCl-containing ETD buffer (pH8.0) to achieve a final salt concentration of 0.5M NaCl, and incubatedovernight at 4° C. with 4 nM [³H]E₂ in the presence or absence of0.1-100 nM unlabeled competitive ligand. Protein-bound [³H]E₂ wasseparated from unbound sterol by incubation with dextran-coatedcharcoal. Experiments were conducted in triplicate.

Example 7 Western Blot Analysis

Denatured cell extracts or purified protein were subjected toelectrophoresis using 4-12% SDS-polyacrylamide gels and transferred tonitrocellulose membranes as described in Chen et al., “Cloning andExpression of a Novel Dominant-Negative-Acting Estrogen ResponseElement-Binding Protein in the Heterogeneous Nuclear RibonucleoproteinFamily,” J Biol Chem, 273: 31352-31357 (1998). The membranes wereblocked with 5% nonfat dry milk for 1 hour and then incubated withmonoclonal anti-human hsp27 antibody (Santa Cruz Biotechnology Inc;Santa Cruz, Calif.; hereinafter “Santa Cruz”) for 2 hours and withHRP-conjugated secondary antibody for another 1 hour prior to detectionof antibody-reactive proteins with chemiluminescence reagent (ECL;Amersham Pharmacia Biotech).

Example 8 Immunoprecipitation

Cells were washed with PBS twice and lysed with RIPA buffer (1×PBScontaining 1% nonidet p-40, 0.5% sodium deoxycholate, 0.1 mM PMSF, 30μl/ml of aprotinin, and 10 mM sodium orthovanadate) (obtained fromSigma-Aldrich Corp.; St. Louis, Mo.) by incubation on ice for 10minutes. The resulting lysates were then disrupted by repeatedaspiration through a 23 gauge needle and cell supernatants obtained bycentrifugation (14,000×g for 10 minutes). Aliquots of supernatant(containing 50 μg protein each) were then incubated with anti-ERα oranti-hsp27 antibody overnight at 4° C. 20 μl of protein A/G agarose(obtained from Santa Cruz) was added and incubated at 4° C. for anotherhour. The protein mixtures were then washed by repeated centrifugationin RIPA buffer (×4) and PBS (×1). The resulting pellet was resuspendedin 2×SDS sample buffer. After boiling, sampled were analyzed by 4-20%SDS-PAGE and separated proteins transferred to nitrocellulose membranes.Western blot analyses were then carried out using anti-ERα andanti-hsp27 antibodies and visualized by ECL (FIG. 5).

Example 9 Yeast Two-Hybrid Screening

The full-length ERα cDNA was amplified using the oligonucleotides5′-GGGGAATTCCATATGACCATGACCCTCCACACCAAAGCATCAGGG-3′ (SEQ ID NO:6) and5′-GCCAGGGGGATCCTCAGACTGTGGCAGGGAAACCCTC-3′ (SEQ ID NO:7). The ERα cDNAwas cloned into the Nde I and Bam HI site of GAL4 DNA binding domainvector (GAL4 DNA-BD/ER). The full length human hsp27 cDNA was amplifiedusing oligonucleotides 5′-GCCGAATTCGCCCAGCGCCCCGCATTTT-3′ (SEQ ID NO:8)and 5′-CCCCTCGAGGGTGGTTGCTTTGAACTTTATTTGAG-3′ (SEQ ID NO:9). The IEBPcDNA was cloned into EcORI and XhoI site of GAL4 DNA activation domainvector. GAL4 DNA-BD/ER was cotransformed with the GAL4 DNA-AD/hsp27plasmid using Yeast Transformation System 2 kit (obtained from Clontech;Palo Alto, Calif.) according to manufacturer's instructions; some of theplates were treated with water (control), E₂ (10-100 nM) or tamoxifen(10-100 nM).

The plasmids were confirmed by automated sequencing. Yeast two-hybridanalysis using the Yeast Two-Hybrid System 3 kit (obtained fromClontech) was performed according to manufacturer's instructions; again,with the exception that the plates were treated with water, E₂ (10-100nM) or tamoxifen (10-100 nM).

Example 10 GST-Pull-Down Assay

A GST-fusion protein with the ligand-binding domain (“LBD”) of ERα(residues 246-595) was expressed in E coli strain DH5α and purified byglutathione sepharose beads according to manufacturer's instructions(Pharmacia Biotech; Piscataway, N.J.). Postnuclear extracts were appliedto the GST beads and incubated for 1 hour. The loaded GST-extractmixture was then washed repetitively (5×) with PBS buffer containing 5mM DTT and 1 mM PMSF and resuspended in 2×SDS sample buffer and boiledfor 5 minutes. Denatured proteins were resolved on 4-20% SDS-PAGE gel,transferred to a nitrocellulose membrane, probed with appropriateantibody (obtained from Santa Cruz) and visualized by ECL.

Example 11 Expression of IEBP

Previously reported purification of IEBP using E₂-affinity columnextractions confirmed its high capacity for estrogen binding but did notclarify the functional relevance of the protein in NWP cells.Experiments were therefore carried out to clarify whether theoverexpression of IEBP antagonized, facilitated, or did nothing toestrogen-induced transactivation. Estrogen-responsive Old World primatecells were transiently co-transfected with IEBP cDNA and anERE-promoter-reporter construct. After transfection, ERE-directedluciferase activity was reduced 50% compared to vector alone-transfectedOld World primate cells. Over-expression of ERE-BP also suppressedERE-mediated transcription, and when IEBP and ERE-BP were co-transfectedthere was an additive decrease in ERE luciferase reporter activity. Theeffect of IEBP was partially abrogated but not restored to normal bypre-treatment with E₂, which also stimulated ERE-mediated transcriptionin control cells. Similar results were also obtained following stabletransfection of IEBP into Old World primate cells. Increased expressionof IEBP in these cells was confirmed by Western blot analyses using ananti-hsp27 antibody. These studies also showed that E₂ stimulated hsp27expression in a dose-dependent fashion in wild-type control cells.Subsequent promoter-reporter data showed that ERE luciferase reporteractivity decreased 2-3 fold in the presence of IEBP when compared withwild-type cells. As with the transient transfectants, this effect wasonly partially abrogated by pre-treatment with E₂. Using the stabletransfectant variants, it was also possible to assess the relationshipbetween IEBP-modulated transcription and postnuclear binding of E₂.

Example 12 Characterization of IEBP

Analysis of both transient and stable transfectants indicated that IEBPacts to squelch ERE-directed gene transcription. This is believed to bedue, at least in part, to enhanced binding of estrogens. To determinewhether IEBP also functions as a direct competitor for ERE-binding, theinventors carried out electrophoretic mobility shift analyses (EMSAs)using an idealized ERE as a probe with recombinant ERα and/orpostnuclear extracts from OWP-IEBP stable transfectant clone 1 asbinding proteins. Data showed that IEBP neither bound to ERE norcompeted with the ERα for binding to ERE. Although IEBP did not appearto bind directly to the ERE, its ability to squelch ER-mediatedtranscription was consistent with possible direct interaction with theERα. To assess this possibility, coimmunoprecipitation was performed byusing anti-ERα and anti-hsp27 antibodies. Antihuman ERα antibodies wereused to immunoprecipitate ERα-untreating proteins in postnuculearextracts of both wild-type and IEBP-stably-transfected cells. Theimmunoprecipitates were subjected to Western blot analysis by usinghsp27 antibodies. Data confirmed association between ERα and hsp27.Similar results were also obtained following initial immunoprecipitationwith hsp27, and subsequent ERα blotting.

Example 13 Ligand-Dependent Interaction of ERα with hsp27

In contrast to the IEBP transfectants, there was relatively littleassociation between ERα and hsp27 in extracts from untreated wild-typecells. However, data indicated the overnight treatment of wild-typecells (but not IEBP transfectants) with E₂ strongly increased hsp27expression, highlighting the possible importance of ligand indetermining ER-Hsp27/IEBP interaction. The yeast two-hybrid system wastherefore employed to confirm ligand-dependent interaction with the ERα.Full-length ERα cDNA was cloned as a fusion protein with the GAL4activation domain, and was used in yeast co-transformations. To identifyproteins that interact with ERα in a ligand specific fashion, yeastcolonies were selected on SD leu-/trp-/His-/Ade-medium supplemented withor without 10 nM E₂ or tamoxifen. The full-length hsp27 and ER insertgrew only in the presence of E₂. No growth was observed in the presenceof the ER antagonist tamoxifen or in selective media without E₂.

Lastly, in order to confirm the interaction between hsp27 and ER,GST-pull-down assays were performed. Protein extracts of cellsoverexpressing IEBP were incubated with a GST-receptor ligand-bindingdomain fusion protein representing the ER. Control assays were employedusing a glucocorticoid receptor (GR)-fusion protein or GST proteinalone. In each case, SDS-PAGE separation that showed the hsp27 wasassessed using anti-hsp27 antibody. Data showed that hsp27 was onlypulled-down by the ER-GST but not GR-GST, or GST alone.

While the description above refers to particular embodiments of thepresent invention, it will be understood that many modifications may bemade without departing from the spirit thereof. The accompanying claimsare intended to cover such modifications as would fall within the truescope and spirit of the present invention. The presently disclosedembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims, rather than the foregoing description,and all changes that come within the meaning and range of equivalency ofthe claims are therefore intended to be embraced therein.

1. An isolated polypeptide, comprising an intracellular estradiolbinding protein (IEBP) as set forth in SEQ ID NO:1.
 2. An isolatedpolypeptide, comprising an intracellular estradiol binding protein(IEBP) that has at least 90% identity to the amino acid sequence setforth in SEQ ID NO:1 wherein the IEBP is capable of binding toestradiol.
 3. The isolated polypeptide of claim 2, wherein the IEBP hasat least 95% identity to the amino acid sequence set forth in SEQ IDNO:
 1. 4. A composition comprising: an isolated polypeptide, comprisingan intracellular estradiol binding protein (IEBP) that has at least 90%identity to the amino acid sequence set forth in SEQ ID NO:1 wherein theIEBP is capable of binding to estradiol; and a carrier.
 5. Thecomposition of claim 4, wherein said isolated polypeptide is an IEBPpolypeptide that has at least 95% identity to the amino acid sequenceset forth in SEQ ID NO:1.
 6. The composition of claim 4, wherein saidisolated polypeptide consists of the sequence set forth in SEQ ID NO:1.7. The composition of claim 4, wherein said carrier is selected from thegroup consisting of phosphate buffered saline (PBS) solution, water,emulsions, and wetting agents, and combinations thereof.